Dominant-negative proteins are capable of inhibiting the binding of nucleic acid binding proteins, i.e., DNA binding proteins, such as transcription regulatory proteins, to target DNA sequences to inactivate gene function. (I. Herskowitz, 1987, Nature, 329:219-222).
The basic-region leucine zipper ("bZIP") DNA binding proteins are a family/class of nucleic acid binding proteins, which are eukaryotic transcription regulatory proteins that regulate transcription of genes by binding as dimers to specific DNA sequences. bZIP proteins characteristically possess two domains--a leucine zipper structural domain and a basic domain that is rich in basic amino acids (C. Vinson et al., 1989, Science, 246:911-916). The two domains are separated by a short segment known as the fork. Two bZIP proteins dimerize by forming a coiled coil region in which the leucine zipper domains dimerize. The basic regions then interact with the major groove of the DNA molecule at a specific DNA sequence site. The binding to DNA stabilizes the dimer. The dimerization and DNA-interaction event regulates eukaryotic gene transcription.
The leucine zipper motif is common to the primary structure of a number of DNA binding proteins, including the yeast transcription factor GCN4, the mammalian transcription factor CCAAT/enhancer-binding protein C/EBP, and the nuclear transforming oncogene products, Fos and Jun, and is characterized by a repeat of leucine amino acids every seven residues (i.e., a heptad repeat); the residues in this region can form amphipathic .alpha.-helices. The leucine-rich amphipathic helices interact and form a dimer complex, called a leucine zipper, at the carboxyl terminus (W. H. Landschultz et al., 1988, Science, 240:1759-1764; A. D. Baxevanis and C. R. Vinson, 1993, Curr. Op. Gen. Devel., 3:278-285), such that the dimerization region forms a coiled coil (E. K. O'Shea et al., 1989, Science, 243:538-542).
Another class of DNA binding proteins, which have similarities to the bZIP motif, are the basic-region helix-loop-helix ("bHLH") proteins (C. Murre et al., 1989, Cell, 56:777-783). bHLH proteins are also composed of discrete domains, the structure of which allows them to recognize and interact with specific sequences of DNA. The helix-loop-helix region promotes dimerization through its amphipathic helices in a fashion analogous to that of the leucine zipper region of the bZIP proteins (R. I. Davis et al., 1990, Cell, 60:733-746; A. Voronova and D. Baltimore, 1990, Proc. Natl. Acad. Sci. USA, 87:4722-4726). Nonlimiting examples of hHLH proteins are myc, max, and mad; myc and mad are known to heterodimerize.
The existence of the leucine zipper in the dimerization region of bZIP proteins allows for a high degree of biological control through the formation of both homodimers and heterodimers. For example, heterodimers are known to form between Fos and Jun (D. Bohmann et al., 1987, Science, 238:1386-1392), among members of the ATF/CREB family (T. Hai et al., 1989, Genes Dev., 3:2083-2090), among members of the C/EBP family (Z. Cao et al., 1991, Genes Dev., 5:1538-1552; S. C. Williams et al., 1991, Genes Dev., 5:1553-1567; and C. Roman et al., 1990, Genes Dev., 4:1404-1415), and between members of the ATF/CREB and Fos/Jun families (T. Hai and T. Curran, 1991, Proc. Natl. Acad. Sci. USA, 88:3720-3724). In general, dimerization of bZIP proteins depends upon the ability of both of the individual carboxyl terminal .alpha.-helices to line up in correct register with one another and to generate a symmetric coiled coil. This, in turn, places the amino terminal basic regions in a symmetric orientation, thus allowing them to interact with DNA (A. D. Baxevanis and C. R. Vinson, 1993, Curr. Op. Gen. Devel., 3:278-285). It has been shown that the ability of the helices within the coiled coil to find the proper register with respect to one another is controlled inherently by the individual helices themselves, and not by the placement of the basic region with respect to the DNA (W. Pu and K. Struhl, 1993, Nucleic Acids Research, 21:4348-4355). However, it will be appreciated that the generation of a symmetric coiled coil structure is not a mandatory requirement for the interaction of the multimerization or dimerization domains of various types of nucleic acid binding proteins.
The bZIP proteins are highly conserved throughout the eukaryotic kingdom and have been isolated and identified in yeast, plants, and mammals. These proteins mediate a variety of biological processes, including oncogenesis, memory, segmentation, and energy regulation (R. Boussoudan, 1994, Cell, 79:59-68; S. Cordes, and G. Barsh, 1994, Cell, 79:1025-1034; S. McKnight et al., 1989, Genes Dev., 3:2021-2024; and I. Verma, 1986, Trends in Genetics, 2:93-96.). Therefore, the ability to inhibit the activity of those proteins associated with oncogenesis or abnormal cell growth and proliferation, for example, is a desirable goal in the field.
In addition, inhibition of the production or function of other cellular proteins that are detrimental, or that influence unwanted or inappropriate phenotypes, in cells, tissues, and, ultimately, the whole organism, is an aim for practitioners in the art.
Of the nearly 70 bZIP proteins that have been identified to date, (H. Hurst, 1994, Protein Profiles, 1:123-168), most can be categorized into one of five major subfamilies on the basis of their DNA recognition properties and amino acid sequence similarities (P. F. Johnson, 1993, Mol. Cell. Biol., 13:6919-6930). These bZIP subgroups include the AP-1, CREB/ATF, C/EBP, PAR, and plant G-box proteins. The proteins in each subfamily recognize highly similar or identical DNA sites whose consensus sequences are 9- or 10-base pair palindromes composed of two 5-base pair half-sites. Binding sites for the various classes of bZIP proteins may differ either by their half-site sequences or their half-site spacing properties. AP-1 proteins, such as Fos, Jun, and GCN4 bind to a 9-base pair pseudopalindromic sequence that can be viewed as two half-sites that overlap by a single base pair, while the consensus binding sites for the other four families have directly abutted pairs of half-sites (N. B. Haas et al., 1995, Mol. Cell. Biol., 15:1923-1932). In addition, thyrotroph embryonic factor (TEF), a transcription factor expressed in the developing anterior pituitary gland, and the liver-enriched albumin D box-binding protein (DBP), (C. R. Mueller et al., 1990, Cell, 61:279-291), have been reported to constitute another class of bZIP proteins (D. W. Drolet et al., 1991, Genes Dev., 5:1739-1753).
bZIP proteins lacking the transactivation domain are naturally occurring dominant negatives that are generally produced by a genetic deletion of the transactivation domain (A. Clark and K. Dougherty, 1993, Biochem J., 296:521-541; P. Descombes and U. Schibler, 1991, Cell, 67:569-579; N. Foulkes et al., 1991, Cell, 64:739-749; and J. Yin et al., 1994, Cell, 79:49-58). These truncated bZIP proteins are able to dimerize and bind to DNA, and if overexpressed, can act as dominant negatives, presumably by competing with the endogenous bZIP protein for its promoter DNA binding site. Accordingly, the truncated bZIP proteins act by mass action to occlude the normal transactivator from the DNA. In addition, it is possible that the deletion of the transactivation domain could also produce a protein having increased, rather than decreased, DNA binding properties. If this were the case, then this type of truncated and naturally occurring dominant negative would not have to be overexpressed to generate particular phenotypes (A. Braiser and A. Kumar, 1994, J. Biol. Chem., 269:10341-10351).
Needed in the art are proteins, expressed and operative in cells, having dominant-negative function to control the transcription of genes or which regulate RNA production and function in a cell. Such expressed proteins can be used for regulating abnormal cell growth in a variety of eukaryotic organisms, including plants, animals, mammals, including humans, insects, microorganisms, and viruses. The present invention provides to the art proteins which can be modified in a particular way to control gene regulation. The particular type of modification may control gene function, for example, to inhibit abnormal or cancer cell growth and proliferation, to inhibit pathogenic diseases caused by microorganisms, particularly eukaryotic microorganisms, such as yeast, and the like, or viruses and may be used as therapeutics for treating pathological diseases and cancer.